The increased incidence of Esophageal Adenocarcinoma (EAC) in the United States over the past two decades represents a significant health challenge. This is especially evident considering the high mortality rate of patients who have undergone treatment for EAC. A current goal of scientific research is to identify molecular markers associated with EAC. Considering the multivariate roles of carbohydrates in cellular processes, one field receiving particular attention is glycomics. A limiting factor for comparative glycomics profiling is the myriad glycan structures postulated to exist in biological samples which present challenges for analytical chemists in the form of component resolution and identification. This is especially problematic for mass spectrometry (MS)-based analytical platforms because isomer resolution cannot be achieved with MS alone. Here we propose the use of ion mobility spectrometry (IMS) techniques combined with MS for the rapid characterization of plasma glycan digests. Specifically, multidimensional IMS (IMS-IMS) methods will be developed to provide the highest efficiency characterization of plasma samples. The combination of IMS-IMS with MS allows for rapid resolution of glycan isomers. This enabling technology allows for high-throughput comparison of hundreds of plasma samples necessary for biomarker validation. As part of the research proposed here, the newly developed technology will be applied to biomarker validation of glycan candidates using a population study of 1000 plasma samples. The work proposed here could have tremendous implications for disease diagnostics as well as the ability to track physiological changes associated with disease progression (or regression resulting from therapy). In addition it is possible that the information-rich datasets will also provide clues into molecular causal mechanisms of disease. PUBLIC HEALTH RELEVANCE: The proposed research will develop ion mobility spectrometry (IMS) techniques coupled with mass spectrometry (MS) for comparative glycomics analyses of human plasma. The new technology provides the greatest ability to resolve glycan isomers and thus correlate changes in specific structures with phenotypic differences. The approach will be used to discover and validate new glycan biomarkers for esophageal adenocarcinoma (EAC).